Polypeptide-human actin-binding protein 54 and a polynucleotide encoding the same

ABSTRACT

The present invention discloses a novel polypeptide—human actin-binding protein 54 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNA recombinant technology. The present invention also discloses methods of using the polypeptide in treatment of various diseases, such as malignant tumor, blood disease, HIV infection, immunological diseases and various inflammation. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. The present invention also discloses the use of such polynucleotide encoding human actin-binding protein 54.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371(c) National Phase of International applicationSerial No. PCT/CN00/00452 filed Nov. 20, 2000, which claims priority toChina patent application Serial No. 99124079.0 filed Nov. 23, 1999, theentire disclosures of which are hereby incorporated in their entireties.

FIELD OF INVENTION

The invention relates to the field of biotechnology. In particular, theinvention relates to a novel polypeptide, human actin-binding protein54, and a polynucleotide sequence encoding said polypeptide. Theinvention also relates to the method for the preparation and use of saidpolynucleotide and polypeptide.

TECHNICAL BACKGROUND

Fascins is a widely distributed protein family. It can concentrateF-actin filaments into bundles. (Edwards, R. A. et al., 1995 Cell Motil.Cytoskeleton 32, 1-9). The genes of fascins have been cloned from human,mice and Xenopus. (Duh, F. M. et al., 1994, DNA Cell Biol. 13:821-827;Edwards, R. A. et al., 1995, J. Biol. Chem. 270, 10764-10770; Holthuis,J. C. M. et al., 1994 Biochim. Biophys. Acta 1219: 184-188). Asactin-binding protein, each fascin is expressed in many cellularstructures such as micropodia and filopodia. The ubiquitous localizationof fascins indicates their important function in morphogenesis ofsubcellular structures. In photoreceptor cells, actin is located ininner segment, synaptic terminal of the rod cells, the connecting ciliumbetween inner segment and outer segment, the labial part of outersegment of rod cells, and the basal part of the rod outer segments.Actin plays an important role in these parts as to regulatemorphogenesis of outer segment disk and in lower vertebrates shorten thephotoreceptor to adjust to brightness (Williams, D. S. et al., 1988, J.Comp. Neurol. 272:161-176). As cell specific actin-binding proteins,fascins can help actin fulfill these functions.

There are three conservative domains in the fascin family of proteins.The largest one consist of twenty eight amino acid residues located inthe N-terminal, and fifteen amino acid residues among these are almostcompletely conserved. The second one is the amino acid sequence ETFQLEin the middle of the full sequence. The third is the sequence GKYW nearthe C-terminal of the proteins.

Sequence analysis of a newly discovered protein reveals that it has allthree conservative domains of the fascin family, with 92% of theconservative amino acids, and it is 95% homologous to human fascin. Sothe protein is a new member of fascin family. And because it isspecifically expressed in photoreceptor cells, it was named retinalfascin (Yoshitsugu, S. et al., 1997 FEBS Lett. 414(2):381-6).

In addition to the three conservative domains, retinal fascin also hasthree highly conservative amino acid of fascin family—Gly 409, Ser 289and Ser 39. Change of the first two amino acid will lead to functionalloss of retinal fascin (Cant, K. et al., 1996, Genetics 143, 249-258).Ser39 is probably a phosphorylation site and phosphorylation of retinalfascin is very important for the organization of microfilaments and cellmobility (Yamakita, Y. et al., 1996, J. Biol. Chem. 271:12632-12638).The full length cDNA of retinal fascin is 1589 bp, including an openreading frame encoding a 492 aa. protein with an apparent molecularweight 55070 Da. There are neither N-terminal signal sequence nortransmembrane domain in retinal fascin.

Besides Ser39, retinal fascin also has phosphorylation sites for proteinkinase A, protein kinase C and tyrosine kinase. These sites are: ninephosphorylation sites for protein kinase C—Thr20, Ser39, Ser147, Thr237,Ser272, The300, Thr375, Thr404, Thr465; and two phosphorylation sitesfor protein kinase A—Thr304, Ser399; two phosphorylation sites forTyrosine kinase—Tyr193, Tyr228. These indicate that retinal fascin isregulated by phosphorylation.

The polypeptide of the present invention is 29% identical and 44%homogenous to retinal fascin at the protein level and has the threeconservative domains of fascin family. So the polypeptide of the presentis named “retinal fascin54” and is found to have similar biologicalfunctions with retinal fascin.

As discussed above, human actin-binding protein p54 plays an essentialrole in the regulation of important biological functions such as celldivision and embryogenesis, and it's believed that many of proteins areinvolved in these regulations. So the determination of those relatedhuman actin-binding proteins, especially of their amino acid sequencesis always desired in this filed. The isolation of this novel humanactin-binding protein p54 builds the basis for research of the proteinfunction under normal and clinical conditions, disease diagnosis anddrug development. So the isolation of its cDNA is very important.

DISCLOSURE OF INVENTION

One objective of the invention is to provide an isolated novelpolypeptide, i.e., a human actin-binding protein 54, and fragments,analogues and derivatives thereof.

Another objective of the invention is to provide a polynucleotideencoding said polypeptide.

Another objective of the invention is to provide a recombinant vectorcontaining a polynucleotide encoding a human actin-binding protein 54.

Another objective of the invention is to provide a geneticallyengineered host cell containing a polynucleotide encoding a humanactin-binding protein 54.

Another objective of the invention is to provide a method for producinga human actin-binding protein 54.

Another objective of the invention is to provide an antibody against ahuman actin-binding protein 54 of the invention.

Another objective of the invention is to provide mimetics, antagonists,agonists, and inhibitors for the polypeptide of the human actin-bindingprotein 54.

Another objective of the invention is to provide a method for thediagnosis and treatment of the diseases associated with an abnormalityof human actin-binding protein 54.

The present invention relates to an isolated polypeptide, which isoriginated from human, and comprises a polypeptide having the amino acidsequence of SEQ ID NO: 2, or its conservative variants, or its activefragments, or its active derivatives and its analogues. Preferably, thepolypeptide has the amino acid sequence of SEQ ID NO: 2.

The present invention also relates to an isolated polynucleotide,comprising a nucleotide sequence or its variant selected from the groupconsisting of (a) the polynucleotide encoding a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; and (b) a polynucleotidecomplementary to the polynucleotide (a); (c) a polynucleotide thatshares at least 70% homology to the polynucleotide (a) or (b).Preferably, said nucleotide sequence is selected from the groupconsisting of (a) the sequence of position 439-1533 in SEQ ID NO: 1; and(b) the sequence of position 1-1704 in SEQ ID NO: 1.

The invention also includes: a vector containing a polynucleotide ofsaid invention, especially an expression vector; a host cell geneticallyengineered with the vector via transformation, transduction ortransfection; a method for the production of said inventive polypeptidethrough the process of host cell cultivation and expression productharvest.

The invention also relates to an antibody which specifically binds tothe inventive polypeptide.

The invention also relates to a method for selecting compounds whichcould simulate, activate, antagonize, or inhibit the activity of theinventive polypeptide and the compounds obtained by the method.

The invention also relates to a method for in vitro diagnosis method ofthe diseases or disease susceptibility related with the abnormalexpression of the inventive polypeptide. The method involves thedetection of mutation in the polypeptide or its encoding polynucleotidesequence, or the determination of its quantity and/or biologicalactivity in biological samples.

The invention also relates to pharmaceutical compositions whichcomprises the inventive polypeptide, its analogues, mimetics, agonists,antagonists, inhibitors, and a pharmaceutically acceptable carrier.

The invention also relates to applications of the inventive polypeptideand/or its polynucleotide for drug development to treat cancers,developmental diseases, immune diseases, or other diseases caused byabnormal expression of the inventive polypeptide.

Other aspects of the invention are apparent to the skilled in the art inview of the disclosure set forth hereinbelow.

The terms used in this specification and claims have the followingmeanings, unless otherwise noted.

“Nucleotide sequence” refers to oligonucleotide, nucleotide, orpolynucleotide and parts of polynucleotide. It also refers to genomic orsynthetic DNA or RNA, which could be single stranded or double stranded,and could represent the sense strand or the antisense strand. Similarly,the term “amino acid sequence” refers to oligopeptide, peptide,polypeptide, or protein sequence and parts of proteins. When the “aminoacid sequence” in the invention is related to the sequence of a naturalprotein, the amino acid sequence of said “peptide” or “protein” will notbe limited to be identical to the sequence of that natural protein.

“Variant” of a protein or polynucleotide refers to the amino acidsequence or nucleotide sequence, respectively with one or more aminoacids or one or more nucleotides changed. Such changes include deletion,insertion, and/or substitution of amino acids in the amino acidsequence, or of nucleotides in the polynucleotide sequence. Thesechanges could be conservative and the substituted amino acid has similarstructural or chemical characteristics as the original one, such as thesubstitution of Ile with Leu. Changes also could be not conservative,such as the substitution of Ala with Trp.

“Deletion” refers to the deletion of one or several amino acids in theamino acid sequence, or of one or several nucleotides in the nucleotidesequence.

“Insertion” or “addition” refers to the addition of one or several aminoacids in the amino acid sequence, or of one or several nucleotides inthe nucleotide sequence, comparing to the natural molecule.“Substitution” refers to the change of one or several amino acids, or ofone or several nucleotides, into different ones without changing numberof the residues.

“Biological activity” refers to structural, regulatory or biochemicalcharacteristics of a natural molecule. Similarly, the term“immungenecity” refers to the ability of natural, recombinant, orsynthetic proteins to inducing a specific immunological reaction, or ofbinding specific antibody in appropriate kind of animal or cell.

“Agonist” refers to molecules which regulate, but generally enhance theactivity of the inventive polypeptide by binding and changing it.Agonists include proteins, nucleotides, carbohydrates or any othermolecules which could bind the inventive polypeptide.

“Antagonist” or “inhibitor” refers to molecules which inhibit or downregulate the biological activity or immunogenicity the inventivepolypeptide via binding to it. Antagonists or inhibitors includeproteins, nucleotides, carbohydrates or any other molecules which bindto the inventive polypeptide.

“Regulation” refers to changes in function of the inventive polypeptide,including up-regulation or down-regulation of the protein activity,changes in binding specificity, changes of any other biologicalcharacteristics, functional or immune characteristics.

“Substantially pure” refers to the condition of substantially free ofother naturally related proteins, lipids, saccharides, or othersubstances. One of ordinary skill in the art can purify the inventivepolypeptide by standard protein purification techniques. Substantiallypure polypeptide of the invention produces a single main band in adenaturing polyacrylamide gel. The purity of a polypeptide may also beanalyzed by amino acid sequence analysis.

“Complementary” or “complementation” refers to the binding ofpolynucleotides by base pairing under the condition of approximate ionconditions and temperature. For instance, the sequence “C-T-G-A” couldbind its complementary sequence “G-A-C-T.” The complementation betweentwo single strand molecules could be partial or complete. Homology orsequence similarity between two single strands obviously influences theefficiency and strength of the formed hybrid.

“Homology” refers to the complementary degree, which may be partially orcompletely homologous. “Partial homology” refers to a sequence beingpartially complementary to a target nucleotide. The sequence could atleast partially inhibit the hybridization between a completelycomplementary sequence and the target nucleotide. Inhibition ofhybridization could be assayed by hybridization (Southern blot orNorthern blot) under less stringent conditions. Substantiallycomplementary sequence or hybrid probe could compete with the completelycomplementary sequence and inhibit its hybridization with the targetsequence under less stringent conditions. This doesn't mean thatnonspecific binding is allowed under a less stringent condition, becausespecific or selective reaction is still required.

“Sequence Identity” refers to the percentage of sequence identity orsimilarity when two or several amino acid or nucleotide sequences arecompared. Sequence identity may be determined by computer programs suchas MEGALIGN (Lasergene Software Package, DNASTAR, Inc., Madison Wis.).MEGALIGN can compare two or several sequences using differentmethodologies such as the Cluster method (Higgins, D. G. and P. M. Sharp(1988) Gene 73:237-244). Cluster method examines the distance betweenall pairs and arrange the sequences into clusters. Then the clusters arepartitioned by pair or group. The sequence identity between two aminoacid sequences such as sequence A and B can be calculated by thefollowing equation:

$\frac{\begin{matrix}{{Number}\mspace{14mu}{of}\mspace{14mu}{paired}\mspace{14mu}{identical}\mspace{14mu}{residues}} \\{{between}{\mspace{11mu}\;}{sequences}\mspace{14mu} A\mspace{14mu}{and}\mspace{14mu} B}\end{matrix}}{\begin{matrix}{{{Residue}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{sequence}\mspace{14mu} A} -} \\{{{number}\mspace{14mu}{of}\mspace{14mu}{gap}\mspace{14mu}{residues}\mspace{14mu}{in}\mspace{14mu}{sequence}\mspace{14mu} A} -} \\{{number}\mspace{14mu}{of}\mspace{14mu}{gap}\mspace{14mu}{residue}\mspace{14mu}{in}\mspace{14mu}{sequence}\mspace{14mu} B}\end{matrix}} \times 100$

Sequence identity between nucleotide sequences can also be determined byCluster method or other well-known methods in the art such as the JotunHein method (Hein J., (1990) Methods in Enzymology 183:625-645)

“Similarity” refers to the degree of identity or conservativesubstitution degree of amino acid residues in corresponding sites of theamino acid sequences when compared to each other. Amino acids forconservative substitution are: negative charged amino acids includingAsp and Glu; positive charged amino acids including Leu, Ile and Val;Gly and Ala; Asn and Gln; Ser and Thr; Phe and Tyr.

“Antisense” refers to the nucleotide sequences complementary to aspecific DNA or RNA sequence. “Antisense strand” refers to thenucleotide strand complementary to the “sense strand.”

“Derivative” refers to the inventive polypeptide or the chemicallymodified nucleotide encoding it. This kind of modified chemical can bederived from replacement of the hydrogen atom with Alkyl, Acyl, orAmino. The nucleotide derivative can encode peptide retaining the majorbiological characteristics of the natural molecule.

“Antibody” refers to the intact antibody or its fragments such as Fa,F(ab′)2 and Fv, and it can specifically bind to antigenic epitopes ofthe inventive polypeptide.

“Humanized antibody” refers to an antibody which has its amino acidsequence in non-antigen binding region replaced to mimic human antibodyand still retain the original binding activity.

The term “isolated” refers to the removal of a material out of itsoriginal environment (for instance, if it's naturally produced, originalenvironment refers to its natural environment). For example, a naturallyproduced polynucleotide or a polypeptide in its original host organismmeans it has not been “isolated,” while the separation of thepolynucleotide or a polypeptide from its coexisting materials in naturalsystem means it was “isolated.” This polynucleotide may be a part of avector, or a part of a compound. Since the vector or compound is notpart of its natural environment, the polynucleotide or peptide is still“isolated.”

As used herein, the term “isolated” refers to a substance which has beenisolated from the original environment. For naturally occurringsubstance, the original environment is the natural environment. Forexample, the polynucleotide and polypeptide in a naturally occurringstate in the viable cells are not isolated or purified. However, if thesame polynucleotide and polypeptide have been isolated from othercomponents naturally accompanying them, they are isolated or purified.

As used herein, “isolated human actin-binding protein 54,” means thathuman actin-binding protein 54 does not essentially contain otherproteins, lipids, carbohydrate or any other substances associatedtherewith in nature. The skilled in the art can purify humanactin-binding protein 54, by standard protein purification techniques.The purified polypeptide forms a single main band on a non-reducing PAGEgel. The purity of human actin-binding protein 54 can also be analyzedby amino acid sequence analysis.

The invention provides a novel polypeptide—human actin-binding protein54, which comprises the amino acid sequence shown in SEQ ID NO: 2. Thepolypeptide of the invention may be a recombinant polypeptide, naturalpolypeptide, or synthetic polypeptide, preferably a recombinantpolypeptide. The polypeptide of the invention may be a purified naturalproduct or a chemically synthetic product. Alternatively, it may beproduced from prokaryotic or eukaryotic hosts, such as bacterial, yeast,higher plant, insect, and mammal cells, using recombinant techniques.Depending on the host used in the protocol of recombinant production,the polypeptide of the invention may be glycosylated ornon-glycosylated. The polypeptide of the invention may or may notcomprise the starting Met residue.

The invention further comprises fragments, derivatives and analogues ofhuman actin-binding protein 54. As used in the invention, the terms“fragment,” “derivative” and “analogue” mean the polypeptide thatessentially retains the same biological functions or activity of humanactin-binding protein 54 of the invention. The fragment, derivative oranalogue of the polypeptide of the invention may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or (ii) one in which one or more of theamino acid residues are substituted with other residues, including asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthe skilled in the art from the teachings herein.

The invention provides an isolated nucleic acid or polynucleotide whichcomprises the polynucleotide encoding an amino acid sequence of SEQ IDNO: 2. The polynucleotide sequence of the invention includes thenucleotide sequence of SEQ ID NO: 1. The polynucleotide of the inventionwas identified in a human embryonic brain cDNA library. Preferably, itcomprises a full-length polynucleotide sequence of 1704 bp, whose ORF(439-1533) encodes 364 amino acids. Based on amino acid homologycomparison, it is found that the encoded polypeptide is 32% homologousto hare R2D2 antigen. This novel human actin-binding protein 54 hassimilar structures and biological functions to those of the hare R2D2antigen.

The polynucleotide according to the invention may be in the forms of DNAor RNA. The forms of DNA include cDNA, genomic DNA, and synthetic DNA,etc., in single stranded or double stranded form. DNA may be an encodingstrand or a non-encoding strand. The coding sequence for maturepolypeptide may be identical to the coding sequence shown in SEQ ID NO:1, or is a degenerate sequence. As used herein, the term “degeneratesequence” means a sequence which encodes a protein or peptide comprisinga sequence of SEQ ID NO: 2 and which has a nucleotide sequence differentfrom the sequence of coding region in SEQ ID NO: 1.

The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2includes those encoding only the mature polypeptide, those encodingmature polypeptide plus various additional coding sequence, the codingsequence for mature polypeptide (and optional additional encodingsequence) plus the non-coding sequence.

The term “polynucleotide encoding the polypeptide” includespolynucleotides encoding said polypeptide and polynucleotides comprisingadditional coding and/or non-coding sequences.

The invention further relates to variants of the above polynucleotideswhich encode a polypeptide having the same amino acid sequence ofinvention, or a fragment, analogue and derivative of said polypeptide.The variant of the polynucleotide may be a naturally occurring allelicvariant or a non-naturally occurring variant. Such nucleotide variantsinclude substitution, deletion, and insertion variants. As known in theart, an allelic variant may have a substitution, deletion, and insertionof one or more nucleotides without substantially changing the functionsof the encoded polypeptide.

The present invention further relates to polynucleotides, whichhybridize to the hereinabove-described sequences, that is, there is atleast 50% and preferably at least 70% identity between the sequences.The present invention particularly relates to polynucleotides, whichhybridize to the polynucleotides of the invention under stringentconditions. As herein used, the term “stringent conditions” means thefollowing conditions: (1) hybridization and washing under low ionicstrength and high temperature, such as 0.2×SSC, 0.1% SDS, 60° C.; or (2)hybridization after adding denaturants, such as 50% (v/v) formamide,0.1% bovine serum/0.1% Ficoll, 42° C.; or (3) hybridization only whenthe homology of two sequences at least 95%, preferably 97%. Further, thepolynucleotides which hybridize to the hereinabove describedpolynucleotides encode a polypeptide which retains the same biologicalfunction and activity as the mature polypeptide of SEQ ID NO: 2

The invention also relates to nucleic acid fragments hybridized with thehereinabove sequence. As used in the present invention, the length ofthe “nucleic acid fragment” is at least more than 10 bp, preferably atleast 20-30 bp, more preferably at least 50-60 bp, and most preferablyat least 100 bp. The nucleic acid fragment can be used in amplificationtechniques of nucleic acid, such as PCR, so as to determine and/orisolate the polynucleotide encoding human actin-binding protein 54.

The polypeptide and polynucleotide of the invention are preferably inthe isolated form, preferably purified to be homogenous.

According to the invention, the specific nucleic acid sequence encodinghuman actin-binding protein 54 can be obtained in various ways. Forexample, the polynucleotide is isolated by hybridization techniqueswell-known in the art, which include, but are not limited to 1) thehybridization between a probe and genomic or cDNA library so as toselect a homologous polynucleotide sequence, and 2) antibody screeningof expression library so as to obtain polynucleotide fragments encodingpolypeptides having common structural features.

According to the invention, DNA fragment sequences may further beobtained by the following methods: 1) isolating double-stranded DNAsequence from genomic DNA; and 2) chemical synthesis of DNA sequence soas to obtain the double-stranded DNA.

Among the above methods, the isolation of genomic DNA is leastfrequently used. A commonly used method is the direct chemical synthesisof DNA sequence. A more frequently used method is the isolation of cDNAsequence. Standard methods for isolating the cDNA of interest is toisolate mRNA from donor cells that highly express said gene followed byreverse transcription of mRNA to form plasmid or phage cDNA library.There are many established techniques for extracting mRNA and the kitsare commercially available (e.g. Qiagene). Conventional method can beused to construct cDNA library (Sambrook, et al., Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). ThecDNA libraries are also commercially available. For example, ClontechLtd. has various cDNA libraries. When PCR is further used, even anextremely small amount of expression products can be cloned.

Numerous well-known methods can be used for screening for thepolynucleotide of the invention from cDNA library. These methodsinclude, but are not limited to, (1) DNA-DNA or DNA-RNA hybridization;(2) the appearance or loss of the function of the marker-gene; (3) thedetermination of the level of human actin-binding protein 54transcripts; (4) the determination of protein product of gene expressionby immunology methods or the biological activity assays. The abovemethods can be used alone or in combination.

In method (1), the probe used in the hybridization could be homologousto any portion of polynucleotide of invention. The length of probe istypically at least 10 nucleotides, preferably at least 30 nucleotides,more preferably at least 50 nucleotides, and most preferably at least100 nucleotides. Furthermore, the length of the probe is usually lessthan 2000 nucleotides, preferably less than 1000 nucleotides. The probeusually is the DNA sequence chemically synthesized on the basis of thesequence information. Of course, the gene of the invention itself or itsfragment can be used as a probe. The labels for DNA probe include, e.g.,radioactive isotopes, fluoresceins or enzymes such as alkalinephosphatase.

In method (4), the detection of the protein products expressed by humanactin-binding protein 54 gene can be carried out by immunology methods,such as Western blotting, radioimmunoassay, and ELISA.

The method of amplification of DNA/RNA by PCR (Saiki, et al. Science1985; 230:1350-1354) is preferably used to obtain the polynucleotide ofthe invention. Especially when it is difficult to obtain the full-lengthcDNA, the method of RACE (RACE-cDNA terminate rapid amplification) ispreferably used. The primers used in PCR can be selected according tothe polynucleotide sequence information of the invention disclosedherein, and can be synthesized by conventional methods. The amplifiedDNA/RNA fragments can be isolated and purified by conventional methodssuch as gel electrophoresis.

Sequencing of polynucleotide sequence of the gene of the invention orits various DNA fragments can be carried out by the conventional dideoxysequencing method (Sanger et al. PNAS, 1977, 74: 5463-5467). Sequencingof polynucleotide sequence can also be carried out using thecommercially available sequencing kits. In order to obtain thefull-length cDNA sequence, it is necessary to repeat the sequencingprocess. Sometimes, it is needed to sequence the cDNA of several clonesto obtain the full-length cDNA sequence.

The invention further relates to a vector comprising the polynucleotideof the invention, a genetically engineered host cell transformed withthe vector of the invention or directly with the sequence encoding humanactin-binding protein 54, and a method for producing the polypeptide ofthe invention by recombinant techniques.

In the present invention, the polynucleotide sequences encoding humanactin-binding protein 54 may be inserted into a vector to form arecombinant vector containing the polynucleotide of the invention. Theterm “vector” refers to a bacterial plasmid, bacteriophage, yeastplasmid, plant virus or mammalian virus such as adenovirus, retrovirusor any other vehicle known in the art. Vectors suitable for use in thepresent invention include, but are not limited to the T7-basedexpression vector for expression in bacteria (Rosenberg, et al., Gene,56:125, 1987), the pMSXND expression vector for expression in mammaliancells (Lee and Nathans, J Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. Any plasmidor vector can be used to construct the recombinant expression vector aslong as it can replicate and is stable in the host. One importantfeature of an expression vector is that the expression vector typicallycontains an origin of replication, a promoter, a marker gene as well astranslation regulatory components.

Methods known in the art can be used to construct an expression vectorcontaining the DNA sequence of human actin-binding protein 54 andappropriate transcription/translation regulatory components. Thesemethods include in vitro recombinant DNA technique, DNA synthesistechnique, in vivo recombinant technique and so on (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989). The DNA sequence is operatively linked to a properpromoter in an expression vector to direct the synthesis of mRNA.Exemplary promoters are lac or trp promoter of E. coli; PL promoter of λphage; eukaryotic promoters including CMV immediate early promoter, HSVthymidine kinase promoter, early and late SV40 promoter, LTRs ofretrovirus, and other known promoters which control gene expression inthe prokaryotic cells, eukaryotic cells or viruses. The expressionvector may further comprise a ribosome binding site for initiatingtranslation, transcription terminator and the like. Transcription inhigher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10 to 300 bp in length that act on a promoter to increase genetranscription level. Examples include the SV40 enhancer on the late sideof the replication origin 100 to 270 bp, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

Further, the expression vector preferably comprises one or moreselective marker genes to provide a phenotype for the selection of thetransformed host cells, e.g., the dehydrofolate reductase, neomycinresistance gene and GFP (green flurencent protein) for eukaryotic cells,as well as tetracycline or ampicillin resistance gene for E. coli.

An ordinarily skilled in the art know clearly how to select appropriatevectors, transcriptional regulatory elements, e.g., promoters,enhancers, and selective marker genes.

According to the invention, polynucleotide encoding human actin-bindingprotein 54 or recombinant vector containing said polynucleotide can betransformed or transfected into host cells to construct geneticallyengineered host cells containing said polynucleotide or said recombinantvector. The term “host cell” means prokaryote, such as bacteria; orprimary eukaryote, such as yeast; or higher eukaryotic, such asmammalian cells. Representative examples are bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;plant cells; insect cells such as Drosophila S2 or Sf9; animal cellssuch as CHO, COS or Bowes melanoma.

Transformation of a host cell with the DNA sequence of invention or arecombinant vector containing said DNA sequence may be carried out byconventional techniques as are well known to those skilled in the art.When the host is prokaryotic, such as E. coli, competent cells, whichare capable of DNA uptake, can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl2 can beused. Transformation can also be carried out by electroporation, ifdesired. When the host is an eukaryote, transfection methods as well ascalcium phosphate precipitation may be used. Conventional mechanicalprocedures such as micro-injection, electroporation, orliposome-mediated transfection may also be used.

The recombinant human actin-binding protein 54 can be expressed orproduced by the conventional recombinant DNA technology (Science, 1984;224:1431), using the polynucleotide sequence of the invention. The stepsgenerally include:

(1) transfecting or transforming the appropriate host cells with thepolynucleotide (or variant) encoding human actin-binding protein 54 ofthe invention or the recombinant expression vector containing saidpolynucleotide;

(2) culturing the host cells in an appropriate medium; and

(3) isolating or purifying the protein from the medium or cells.

In Step (2) above, depending on the host cells used, the medium forcultivation can be selected from various conventional mediums. The hostcells are cultured under a condition suitable for its growth until thehost cells grow to an appropriate cell density. Then, the selectedpromoter is induced by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.

In Step (3), the recombinant polypeptide may be included in the cells,or expressed on the cell membrane, or secreted out of the cell. Ifdesired, physical, chemical and other properties can be utilized invarious isolation methods to isolate and purify the recombinant protein.These methods are well-known to those skilled in the art and include,but are not limited to conventional renaturation treatment, treatment bya protein precipitant (such as salt precipitation), centrifugation, celllysis by osmosis, sonication, supercentrifugation, molecular sievechromatography or gel chromatography, adsorption chromatography, ionexchange chromatography, HPLC, and any other liquid chromatography, anda combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are provided to illustrate the embodiment of theinvention, not to limit the scope of invention defined by the claims.

FIG. 1 shows an alignment comparison of amino acid sequences of humanactin-binding protein 54 of the invention and retinal fascin. The uppersequence is human actin-binding protein 54, and the lower sequence isRetinal fascin. The identical and similar amino acids are indicated by aone-letter code of amino acid and “+” respectively.

FIG. 2 shows the SDS-PAGE of the isolated human actin-binding protein54, which has a molecular weight of 54 kDa. The isolated protein band ismarked with an arrow.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention is further illustrated by the following examples. It isappreciated that these examples are only intended to illustrate theinvention, not to limit the scope of the invention. For the experimentalmethods in the following examples, they are performed under routineconditions, e.g., those described by Sambrook. et al., in MoleculeClone: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress, 1989, or as instructed by the manufacturers, unless otherwisespecified.

EXAMPLE 1 Cloning of Human Actin-Binding Protein 54 Gene

Total RNA from a human embryonic brain was extracted by the one-stepmethod with guanidinium isocyanate/phenol/chloroform. The poly(A) mRNAwas isolated from the total RNA with Quik mRNA Isolation Kit (Qiegene).cDNA was prepared by reverse transcription with 2 μg poly(A) mRNA. ThecDNA fragments were inserted into the polyclonal site of pBSK(+) vector(Clontech) using Smart cDNA cloning kit (Clontech) and then transformedinto DH5α to form the cDNA library. The 5′- and 3′-ends of all cloneswere sequenced with Dye terminate cycle reaction sequencing kit(Perkin-Elmer) and ABI 377 Automatic Sequencer (Perkin-Elmer). Thesequenced cDNA were compared with the public database of DNA sequences(Genebank) and the DNA sequence of one clone 1030c03 was found to be anovel DNA sequence. The inserted cDNA sequence of clone 1030c03 wasdual-directionally sequenced with a serial of synthesized primers. Itwas indicated that the full length cDNA contained in clone 1030c03 was1704 bp (SEQ ID NO: 1) with a 1094 bp ORF located in positions 439-1533which encoded a novel protein (SEQ ID NO: 2). This clone was namedpBS-1030c03 and the encoded protein was named human actin-bindingprotein 54.

EXAMPLE 2 Homology Search of cDNA Clone

The homology research of the DNA sequence and its protein sequence ofhuman actin-binding protein 54 of the invention were performed by Blast(Basic local Alignment search tool) (Altschul, SF et al. J. Mol. Biol.1990; 215:403-10) in databases such as Genbank, Swissport, etc. The mosthomologous gene to human actin-binding protein 54 of the invention isknown Retinal Fascin. The Genbank accession number of its encodedprotein is AB003485. The alignment result of the protein was shown inFIG. 1. Two proteins are highly homologous with an identity of 29% and asimilarity of 44%.

EXAMPLE 3 Cloning Human Actin-Binding Protein 54 Gene by RT-PCR

The template was total RNA extracted from a human embryonic brain. Thereverse transcription was carried out with oligo-dT primer to producecDNAs. After cDNA purified with Qiagen Kit, PCR was carried out with thefollowing primers:

Primer 1: 5′-GAGGGGATAGTGGGAAAGTGGAA-3′ (SEQ ID NO: 3)

Primer 2: 5′-GGGTGGCCTGTTGTGATGTATTG-3′ (SEQ ID NO: 4)

Primer 1 is the forward sequence started from position 1 of 5′ end ofSEQ ID NO: 1.

Primer 2 is the reverse sequence of the 3′ end of SEQ ID NO: 1.

The amplification condition was a 50 ul reaction system containing 50mmol/L KCl, 10 mmol/L Tris-Cl (pH 8.5), 1.5 mmol/L MgCl₂, 200 mmol/LdNTP, 10 pmol of each primer, 1U Taq DNA polymerase(Clontech). Thereaction was performed on a PE 9600 DNA amplifier with the followingparameters: 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min for 25cycles. β-actin was used as a positive control, and a blank template, asa negative control in RT-PCR. The amplified products were purified witha QIAGEN kit, and linked with a pCR vector (Invitrogen) using a TACloning Kit. DNA sequencing results show that the DNA sequence of PCRproducts was identical to nucleotides 1-1704 bp of SEQ ID NO: 1.

EXAMPLE 4 Northern Blotting of Expression of Human Actin-Binding Protein54 Gene

Total RNA was extracted by one-step method (Anal. Biochem 1987, 162,156-159) with guanidinium isocyanate-phenol-chloroform. That is,homogenate the organize using 4M guanidinium isocyanate-25 mM sodiumcitrate, 0.2 sodium acetate(pH 4.0), add 1 volume phenol and ⅕ volumechloroform-isoamyl alcohol(49:1), centrifuge after mixing. Take out thewater phase, add 0.8 volume isopropyl alcohol, then centrifuge themixture. Wash the RNA precipitation using 70% ethanol, then dry, thendissolve it in the water. 20 μg RNA was electrophoresed on the 1.2%agarose gel containing 20 mM 3-(N-morpholino) propane sulfonic acid(pH7.0)-5 mM sodium acetate-imM EDTA-2.2M formaldehyde. Then transfer it toa nitrocellulose filter. Prepare the 32P-labelled DNA probe with α-³²PdATP by random primer method. The used DNA probe is the coding sequence(439 bp-1533 bp) of human actin-binding protein 54 amplified by PCRindicated in FIG. 1. The nitrocellulose filter with the transferred RNAwas hybridized with the ³²P-labelled DNA probe (2×10⁶ cpm/ml) overnightin a buffer containing 50% formamide-25 mM KH₂PO₄(Ph7.4)-5× Denhardt'ssolution and 200 μg/ml salmine. Then wash the filter in the 1×SSC-0.1%SDS, at 55° C., for 30 min. Then analyze and quantitative determinateusing Phosphor Imager.

EXAMPLE 5 In Vitro Expression, Isolation and Purification of RecombinantHuman Actin-Binding Protein 54

A pair of primers for specific amplification was designed based on SEQID NO: 1 and the encoding region in FIG. 1, the sequences are asfollows:

Primer 3:5′-CCCCATATGATGTGGACCCCCCGACCAGCCCTCC-3′(SEQ ID NO: 5)

Primer 4: 5′-CCCGAATTCCTAAAATTCCCAGATACACTCTCTG-3′(SEQ ID NO: 6)

These two primers contain a NdeI and EcoRI cleavage site on the 5′ endrespectively. Within the sites are the coding sequences of the 5′ and 3′end of the desired gene. NdeI and EcoRI cleavage sites werecorresponding to the selective cleavage sites on the expression vectorpET-28b(+) (Novagen, Cat. No. 69865.3). PCR amplification was performedwith the plasmid pBS-1030c03 containing the full-length target gene as atemplate. The PCR reaction was subject to a 50 μl system containing 10pg pBS-1030c03 plasmid, 10 pmol of Primer-3 and 10 pmol of Primer-4, 1μl of Advantage polymerase Mix (Clontech). The parameters of PCR were94° C. 20 sec, 60° C. 30 sec, and 68° C. 2 min for 25 cycles. Afterdigesting the amplification products and the plasmid pET-28(+) by NdeIand EcoRI, the large fragments were recovered and ligated with T4ligase. The ligated product was transformed into E. coli DH5α with thecalcium chloride method. After cultured overnight on a LB platecontaining a final concentration of 30 μg/ml kanamycin, positive cloneswere selected out using colony PCR and then sequenced. The positiveclone (pET-1030c03) with the correct sequence was selected out and therecombinant plasmid thereof was transformed into BL21(DE3)plySs(Novagen) using the calcium chloride method. In a LB liquid mediumcontaining a final concentration of 30 μg/ml of kanamycin, the hostbacteria BL21(pET-1030c03) were cultured at 37° C. to the exponentialgrowth phase, then IPTG were added with the final concentration of 1mmol/L, the cells were cultured for another 5 hours, and thencentrifuged to harvest the bacteria. After the bacteria were sonicated,the supernatant was collected by centrifugation. Then the purifieddesired protein—human actin-binding protein 54 was obtained by aHis.Bind Quick Cartridge (Novagen) affinity column with binding6His-Tag. SDS-PAGE showed a single band at 54 kDa (FIG. 2). The band wastransferred onto the PVDF membrane and the N terminal amino acid wassequenced by Edams Hydrolysis, which shows that the first 15 amino acidson N-terminus were identical to those in SEQ ID NO: 2.

EXAMPLE 6 Preparation of Antibody Against Human Actin-Binding Protein 54

The following specific human actin-binding protein 54 polypeptide wassynthesized by a polypeptide synthesizer (PE-ABI):NH₂-Met-Trp-Thr-Pro-Arg-Pro-Ala-Leu-His-Val-His-Val-Ile-Leu-Tyr-COOH(SEQ ID NO:7). The polypeptide was conjugated with hemocyanin and bovineserum albumin (BSA) respectively to form two composites (See Avrameas etal., Immunochemistry, 1969, 6:43). 4 mg of hemocyanin-polypeptidecomposite was used to immunize rabbit together with Freund's completeadjuvant. The rabbit was re-immunized with the hemocyanin-polypeptidecomposite and Freund's incomplete adjuvant 15 days later. The titer ofantibody in the rabbit sera was determined with a titration plate coatedwith 15 μg/ml BSA-polypeptide composite by ELISA. The total IgG wasisolated from the sera of an antibody positive rabbit with ProteinA-Sepharose. The polypeptide was bound to Sepharose 4B column activatedby cyanogen bromide. The antibodies against the polypeptide wereisolated from the total IgG by affinity chromatography. Theimmunoprecipitation approved that the purified antibodies couldspecifically bind to human actin-binding protein 54.

EXAMPLE 7 Application of the Polynucleotide Fragments of the Inventionas Hybridization Probes

Oligonucleotides probes selected from the polynucleotide of theinvention have many applications. The probe could be used to determinethe existence of polynucleotide of the invention or its homologouspolynucleotide sequences by hybridization with genomic, or cDNA libraryof normal or clinical tissues from varied sources. The probes could befurther used to determine whether polynucleotide of the invention or itshomologous polynucleotide sequences are abnormally expressed in cellsfrom normal or clinical tissues.

The purpose of the following example is to select suitableoligonucletide fragments from SEQ ID NO:1 as hybridization probes toapply in membrane hybridization to determine whether there ispolynucleotide of the invention or its homologous polynucleotidesequences in sample tissues. Membrane hybridization methods include dotblot, Southern blot, Northern blot, and replica hybridization. Allmethods follow nearly the same steps after the polynucleotide samplesare immobilized on membranes. These steps are: membranes withimmobilized samples are prehybridized in hybridization buffer notcontaining probes to block nonspecific binding sites of the membranes.Then the prehybridization buffer is replaced by hybridization buffercontaining labeled probes and incubation is carried out at theappropriate temperature so probes hybridize with the target nucleotides.Free probes are washed off by a series of washing steps after thehybridization step. A high-stringency washing condition (relatively lowsalt concentration and high temperature) is applied in the example toreduce background and retain highly specific signals. Two types ofprobes are selected for the example: the first type of probes areoligonucleotides identical or annealed to SEQ ID NO:1; the second typeprobes are oligonucleotides partially identical or partially annealed toSEQ ID NO:1. Dot blot method is applied in the example forimmobilization of the samples on membrane. The strongest specific signalproduced by hybridization between first type probes and samples isobtained after relatively strict membrane washing steps.

Selection of Probes

The principles below should be followed for the selection ofoligonucleotide fragments from SEQ ID NO:1 as hybrid probes:

1. The optimal length of probes should be between eighteen and fiftynucleotides.

2. GC amount should be between 30% and 70%, since nonspecifichybridization increases when GC amount is more than 70%.

3. There should be no complementary regions within the probesthemselves.

4. Probes meeting the requirements above could be initially selected forfurther computer-aided sequence analysis, which includes homologycomparison between the initially selected probes and its source sequenceregion (SEQ ID NO: 1), other known genomic sequences and theircomplements. Generally, the initial selected probes should not be usedwhen they share fifteen identical continuous base pairs, or 85% homologywith non-target region.

5. Whether the initially selected probes should be chosen for finalapplication depends upon further experimental confirmation.

The following two probes could be selected and synthesized after theanalysis above:

Probe One belongs to the first type, which is completely identical orannealed to the gene fragments of SEQ ID NO: 1 (41 nucleotides).

Probe Two belongs to the second type which is a substituted or mutantsequence of a fragment of SEQ ID NO: 1 (41 nucleotides).

Any other frequently used reagents unlisted but involved in thefollowing experimental steps and their preparation methods can be found,for example, in: DNA PROBES G. H. Keller; M. M. Manak; Stockton Press,1989 (USA) or a more commonly used molecular cloning experimentalhandbook Molecular Cloning (J. Sambrook et al. Academic press, 1998, 2ndedition).

Sample Preparation:

1. DNA Extraction from Fresh or Frozen Tissues

Steps: 1) Place fresh or newly thawed tissue onto a dish on icecontaining phosphate-buffered saline (PBS). Cut the tissue into smallpieces with scissors or an operating knife. Tissues should be kept dampthrough the operation. 2) Mince the tissue by centrifugation at 2,000 gfor 10 minutes. 3) Re-suspend the pellet (about 10 ml/g) with coldhomogenating buffer (0.25 mol/l saccharose; 25 mmol/l Tris-HCl, pH7.5;25 m mol/LnaCl; 25 mmol/L MgCl2) at 4° C., and homogenate tissuesuspension at full speed with an electronic homogenizer until it'scompletely smashed. 5) Centrifuge at 1,000 g for 10 minutes. 6)Re-suspend the cell pellet (1-5 ml per 0.1 g initial tissue sample), andcentrifuge at 1,000 g for 10 minutes. 7) Re-suspend the pellet withlysis buffer (1-5 ml per 0.1 g initial tissue sample), and continue onto the phenol extraction method.

2. Phenol Extraction of DNA

Steps: 1) Wash cells with 1-10 ml cold PBS buffer and centrifuge at 1000g for 10 minutes. 2) Re-suspend the precipitated cells with at least 100μl cold cell lysis buffer (1×10⁸ cells/ml). 3) Add SDS to a finalconcentration of 1%. Addition of SDS into the cell precipitation beforecell re-suspension will cause the formation of large balls by cellswhich is difficult to be smashed and total production will be reduced.This is especially important when extracting more than 10⁷ cells. 5)Incubate at 50° C. for an hour or shake gently overnight at 37° C. 6)Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) tothe DNA solution to be purified in a microcentrifuge tube, andcentrifuge for 10 minutes. If the two phases are not clearly separated,the solution should be recentrifuged. 7) remove the water phase to a newtube. 8) add an equal volume of chloroform: isoamyl alcohol (24:1) andcentrifuge for 10 minutes. 9) remove the water phase containing DNA to anew tube and then purify DNA by ethanol precipitation.

3. DNA Purification By Ethanol Precipitation

Steps: 1) Add 1/10 vol of 2 mol/L sodium acetate and 2 vol of cold 100%ethanol into the DNA solution, mix and place at −20° C. for an hour orovernight. 2) Centrifuge for 10 minutes. 3) Carefully remove theethanol. 4) Add 500 μl of cold 70% ethanol to wash the pellet andcentrifuge for 5 minutes. 6) Carefully remove the ethanol and invert thetube on absorbent paper to remove remnant ethanol. Air dry for 10-15minutes to evaporate the ethanol on the pellet surface. Do not dry thepellet completely since completely dry pellet is difficult to bedissolved again. 7) Re-suspend the DNA pellet with a small volume of TEor water. Spin at low speed or blow with a pipette, and add TE graduallyand mix until DNA is completely dissolved. About 1 μl of DNA solution isobtained per 1˜5×10⁶ cells.

The following steps 8-13 are applied only when contamination must beremoved, otherwise go directly to step 14. 8) Add Rnase A into DNAsolution to a final concentration of 100 μg/ml and incubate at 37° C.for 30 minutes. 9) Add SDS and protease K to the final concentration of0.5% and 100 μg/ml individually, and incubate at 37° C. for 30 minutes.10) Add an equal volume of phenol: chloroform: isoamyl alcohol(25:24:1), and centrifuge for 10 minutes. 11) Carefully remove the waterphase and extract it with an equal volume of chloroform: isoamyl alcohol(24:1) and centrifuge for 10 minutes. 12) Carefully remove out the waterphase, and add 1/10 vol of 2 mol/L sodium acetate and 2.5 vol of cold100% ethanol, then mix and place at −20° C. for an hour. 13) Wash thepellet with 70% ethanol and 100% ethanol, air dry and re-suspend DNA assame as the steps 3-6. 14) Determine the purity and production of DNA byA260 and A280 assay. 15) Separate DNA sample into several portions andstore at −20° C.

Preparation of Sample Membrane

1) Take 4×2 pieces of nitrocellulose membrane (NC membrane) of desiredsize, and lightly mark out the sample dot sites and sample number with apencil. Every probe needs two pieces of NC membrane, so then membranescould be washed under high stringency condition and moderate stringencycondition individually in the following experimental steps.

2) Pipette 15 μl of samples and control individually, dot them on themembrane, and dry at room temperature.

3) Place the membranes on filter paper soaked in 0.1 mol/LnaOH, 1.5mol/L NaCl, leave for 5 minutes (twice), and allow to dry. Transfer themembranes on filter paper soaked in 0.5 mol/L Tris-HCl (pH7.0), 3 mol/LNaCl, leave for 5 minutes (twice), and allow to dry.

4) place the membranes between clean filter paper, packet with aluminumfoil, and vacuum dry at 60-80° C. for 2 hours.

Labeling of Probes

1) Add 3 μl probe (0.1 OD/10 μl), 2 μl kinase buffer, 8-10 uCiγ-32P-dATP+2U Kinase, and add water to the final volume of 20 μl.

2) Incubate at 37° C. for 2 hours.

3) Add ⅕ vol bromophenol blue indicator (BPB).

4) Load that sample on Sephadex G-50 column.

5) Collect the first peak before the elution of 32P-Probe (monitor theeluting process by Monitor).

6) Five drops each tube and collect for 10-15 tubes.

7) Measure the isotope amount with liquid scintillator.

8) Merged collection of the first peak is the prepared 32P-Probe (thesecond peak is free γ-32P-dATP).

Prehybridization

Place the sample membranes in a plastic bag, add 3-10 mg prehybridbuffer (10× Denhardt, s; 6×SSC, 0.1 mg/ml CT DNA (calf thymus glandDNA)), seal the bag, and shake on a 68° C. water bath for two hourshybridization.

Cut off a corner of the plastic bag, add in prepared probes, seal thebag, and shake on a 42° C. water bath overnight.

Membrane Washing

Membrane washing applying a high-stringency condition:

1) Take out the hybridized sample membranes

2) Wash the membranes with 2×SSC, 0.1% SDS at 40° C. for 15 minutes(twice).

3) Wash the membranes with 0.1×SSC, 0.1% SDS at 40° C. for 15 minutes(twice).

4) Wash the membranes with 0.1×SSC, 0.1% SDS at 55° C. for 30 minutes(twice), and dry at room temperature.

Membrane washing applying a low-stringency condition:

1) Take out the hybridized sample membranes.

2) Wash the membranes with 2×SSC, 0.1% SDS at 37° C. for 15 minutes(twice).

3) Wash the membranes with 0.1×SSC, 0.1% SDS at 37° C. for 15 minutes(twice).

4) Wash the membranes with 0.1×SSC, 0.1% SDS at 40° C. for 15 minutes(twice), and dry at room temperature.

X Ray Autoradiography:

X ray autoradiograph at −70° C. (autoradiograph time varies according toradioactivity of the hybrid spots).

Experimental Results:

In hybridization experiments carried out under low-stringency membranewashing condition, the radioactivity of all the above four probeshybridization spots show no obvious difference; while in hybridizationexperiments carried out under high-stringency membrane washingcondition, radioactivity of the hybridization spot by Probe One isobviously stronger than the other three. So Probe One could be appliedin qualitative and quantitative analysis of the existence anddifferential expression of the polynucleotide of the invention indifferent tissues.

INDUSTRIAL APPLICABILITY

The polypeptide of the invention and antagonists, agonists andinhibitors thereof can be directly used for the treatment of diseases,e.g., various malignant tumors or cancers, dermatitis, inflammation,adrenoprival disease and HIV infection and immune system diseases.

Retinal fascin is mainly expressed in eyes and its abnormal expressionwill cause a series of eye diseases. Meanwhile, as retinal fascin alsois expressed in organs such as brain, heart, bones, muscle, intestine,and lung, its abnormal expression will lead to lesion of these organs,even causing cancers. Retinal fascin is also involved in signaltransduction and regulation of development. The abnormal expression ofhuman actin-binding protein P54 of the invention is concerned, willcause diseases including the following: retinitis, retinal pigmentation,bifid spine, cranioschisis, anencephadly, encephalocele, schizencephalicporencephaly, Down syndrome, congenital hydrocephalus, Aqueductdeformity, achondroplastic dwarf, spine epiphysis dysplasia, pseudoachondroplasia, Langer-Giedion syndrome, funnel chest, gonadaldysgenesis, Congenital adrenal hyperplasia, epispadia; Anaspadias,deformation syndromes accompanied by Microsomia such as Conradi syndromeand Danbolt-Closs syndrome, congenital glaucoma or cataract, congenitalcrystalline lens position abnormality, Congenital small palpebralfissure, retina dysplasia, Congenital optic atrophy, Congenital sensorynerve hearing loss, acrorhagadia, teratosis, Willams syndrome, Alagillesyndrome, Bechwith-Wiedemann syndrome, Transient ischemic attacks,cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage,gliocytoma, meningioma, fibroneuroma, pituitary adenoma, intercraniumgranuloma, reflux esophagitis, esophageal carcinoma, gastritis (acutegastritis, chronic gastritis, other unusual gastritis such as acutecorrosive gastritis, acute phlegmonous gastritis, Gastritis hypertrophicgigantica), peptic ulcer, gastric carcinoma, intestinal tuberculosis,CROHN disease, ulcerative colon, large intestines cancer,gastrointestinal neurosis (globus hystericus, psyche emesis, aerophagia,anorexia nervosa, irritable bowel syndrome), acute diarrhea, chronichepatitis (Chronic persistent hepatitis, chronic active hepatitis),cirrhosis of liver (primary biliary cirrhosis), primary liver carcinoma,hepatic coma, pancreatitis (Acute pancreatitis, chronic pancreatitis),pancreatic carcinoma, tuberculous peritonitis, Upper digestive tractMassive bleeding, salivary gland tumor, esophageal carcinoma, esophagealleiomyosarcoma, primary oesophagus oat-cell carcinoma, gastriccarcinoma, gastric malignant lymphoma, gastric carcinoid, cancer ofcolon, Intestinal malignant lymphoma, primary liver carcinoma, liverblastoma, primary carcinoma of gallbladder, pancreatic carcinoma, acutecardiac insufficiency, Chronic heart failure, sinus tachycardia, sinusbradycardia, sinus arrest, sinoatrial block, SSS, atrial prematurebeats, atrial tachycardia, atrial flutter, auricular fibrillation,atrio-ventricular junctional premature beat, atrio-ventricularjunctional escape beat and cardiac rhythm, noparoxysmal supraventriculartachycardia, paroxysmal supraventricular tachycardia, WPW syndrome,ventricular premature beat, ventricular tachycardia, ventricularflutter, ventricular fibrillation, Atrioventricular block,intraventricular block, isolated pulmonic stenosis, aortic stenosis,coarctation of aorta, idiopathic dilatation of the pulmonary artery,primary pulmonary hypertension, persistent left superior vena cava,dextrocardia, primary hypertension, secondary hypertension,atherosclerosis, CAD (angina pectoris, myocardial infarction, latentcoronary heart disease, cardiac insufficiency, arrhythmia coronary heartdisease, and coronary heart disease of sudden death form), mitralstenosis, mitral incompetence, aortic stenosis, aortic incompetence,tricuspid stenosis, tricuspid incompetence, valvular pulmonary stenosis,pulmonary insufficiency, multiple valve disease, self-valveendocarditis, prosthetic valve endocarditis and vein drug addict'sendocarditis, dilated cardiomyopathy, hypertrophic cardiomyopathy,Restrictive cardiomyopathy, myocarditis, alcoholic heart disease,perinatal heart disease, drug-induced heart disease, Keshan disease,Acute pericarditis, constrictive pericarditis, syphilitis cardiovasculardisease, Takayasu arteritis, emphraxis milieu atherosclerosis, RAYNAUDsyndrome, thrombophlebitis.

The invention also provides methods for screening compounds so as toidentify an agent which enhances human actin-binding protein 54 activity(agonists) or decrease human actin-binding protein 54 activity(antagonists). The agonists enhance the biological functions of humanactin-binding protein 54 such as inactivation of cell proliferation,while the antagonists prevent and cure the disorders associated with theexcess cell proliferation, such as various cancers. For example, in thepresence of an agent, the mammal cells or the membrane preparationexpressing human actin-binding protein 54 can be incubated with thelabeled human actin-binding protein 54 to determine the ability of theagent to enhance or repress the interaction.

Antagonists of human actin-binding protein 54 include antibodies,compounds, receptor deletants and analogues. The antagonists of humanactin-binding protein 54 can bind to human actin-binding protein 54 andeliminate or reduce its function, or inhibit the production of humanactin-binding protein 54, or bind to the active site of said polypeptideso that the polypeptide can not function biologically.

When screening for compounds as an antagonist, human actin-bindingprotein 54 may be added into a biological assay. It can be determinedwhether the compound is an antagonist or not by determining its effecton the interaction between human actin-binding protein 54 and itsreceptor. Using the same method as that for screening compounds,receptor deletants and analogues acting as antagonists can be selected.Polypeptide molecules capable of binding to human actin-binding protein54 can be obtained by screening a polypeptide library comprising variouscombinations of amino acids bound onto a solid matrix. Usually, humanactin-binding protein 54 is labeled in the screening.

The invention further provides a method for producing antibodies usingthe polypeptide, and its fragment, derivative, analogue or cells as anantigen. These antibodies may be polyclonal or monoclonal antibodies.The invention also provides antibodies against epitopes of humanactin-binding protein 54. These antibodies include, but are not limitedto, polyclonal antibody, monoclonal antibody, chimeric antibody,single-chain antibody, Fab fragment and the fragments produced by a Fabexpression library.

Polyclonal antibodies can be prepared by immunizing animals, such asrabbit, mouse, and rat, with human actin-binding protein 54. Variousadjuvants, including but are not limited to Freund's adjuvant, can beused to enhance the immunization. The techniques for producing humanactin-binding protein 54 monoclonal antibodies include, but are notlimited to, the hybridoma technique (Kohler and Milstein. Nature, 1975,256:495-497), the trioma technique, the human B-cell hybridomatechnique, the EBV-hybridoma technique and so on. A chimeric antibodycomprising a constant region of human origin and a variable region ofnon-human origin can be produced using methods well-known in the art(Morrison et al, PNAS, 1985, 81:6851). Furthermore, techniques forproducing a single-chain antibody (U.S. Pat. No. 4,946,778) are alsouseful for preparing single-chain antibodies against human actin-bindingprotein 54.

The antibody against human actin-binding protein 54 can be used inimmunohistochemical method to detect the presence of human actin-bindingprotein 54 in a biopsy specimen.

The monoclonal antibody specific to human actin-binding protein 54 canbe labeled by radioactive isotopes, and injected into human body totrace the location and distribution of human actin-binding protein 54.This radioactively labeled antibody can be used in the non-woundingdiagnostic method for the determination of tumor location andmetastasis.

Antibodies can also be designed as an immunotoxin targeting a particularsite in the body. For example, a monoclonal antibody having highaffinity to human actin-binding protein 54 can be covalently bound tobacterial or plant toxins, such as diphtheria toxin, ricin, ormosine.One common method is to challenge the amino group on the antibody withsulfhydryl cross-linking agents, such as SPDP, and bind the toxin ontothe antibody by interchanging the disulfide bonds. This hybrid antibodycan be used to kill human actin-binding protein 54-positive cells.

The antibody of the invention is useful for the therapy or theprophylaxis of disorders related to the human actin-binding protein 54.The appropriate amount of antibody can be administrated to stimulate orblock the production or activity of human actin-binding protein 54.

The invention further provides diagnostic assays for quantitative and insitu measurement of human actin-binding protein 54 level. These assaysare well known in the art and include FISH assay and radioimmunoassay.The level of human actin-binding protein 54 detected in the assay can beused to illustrate the importance of human actin-binding protein 54 indiseases and to determine the diseases associated with humanactin-binding protein 54.

The polypeptide of the invention is useful in the analysis ofpolypeptide profile. For example, the polypeptide can be specificallydigested by physical, chemical, or enzymatic means, and then analyzed byone, two or three dimensional gel electrophoresis, preferably byspectrometry.

New human actin-binding protein 54 polynucleotides also have manytherapeutic applications. Gene therapy technology can be used in thetherapy of abnormal cell proliferation, development or metabolism, whichare caused by the loss of human actin-binding protein 54 expression orthe abnormal or non-active expression of human actin-binding protein 54.Recombinant gene therapy vectors, such as virus vectors, can be designedto express mutated human actin-binding protein 54 so as to inhibit theactivity of endogenous human actin-binding protein 54. For example, oneform of mutated human actin-binding protein 54 is a truncated humanactin-binding protein 54 whose signal transduction domain is deleted.Therefore, this mutated human actin-binding protein 54 can bind thedownstream substrate without the activity of signal transduction. Thus,the recombinant gene therapy vectors can be used to cure diseases causedby abnormal expression or activity of human actin-binding protein 54.The expression vectors derived from a virus, such as retrovirus,adenovirus, adeno-associated virus, herpes simplex virus, parvovirus,and so on, can be used to introduce the human actin-binding protein 54gene into the cells. The methods for constructing a recombinant virusvector harboring human actin-binding protein 54 gene are described inthe literature (Sambrook, et al. supra). In addition, the recombinanthuman actin-binding protein 54 gene can be packed into liposome and thentransferred into the cells.

The methods for introducing the polynucleotides into tissues or cellsinclude directly injecting the polynucleotides into tissue in the body;or introducing the polynucleotides into cells in vitro with vectors,such as virus, phage, or plasmid, etc, and then transplanting the cellsinto the body.

Also included in the invention are ribozyme and the oligonucleotides,including antisense RNA and DNA, which inhibit the translation of thehuman actin-binding protein 54 mRNA. Ribozyme is an enzyme-like RNAmolecule capable of specifically cutting certain RNA. The mechanism isnucleic acid endo-cleavage following specific hybridization of ribozymemolecule and the complementary target RNA. Antisense RNA and DNA as wellas ribozyme can be prepared by using any conventional techniques for RNAand DNA synthesis, e.g., the widely used solid phase phosphite chemicalmethod for oligonucleotide synthesis. Antisense RNA molecule can beobtained by the in vivo or in vitro transcription of the DNA sequenceencoding said RNA, wherein said DNA sequence is integrated into thevector and downstream of the RNA polymerase promoter. In order toincrease its stability, a nucleic acid molecule can be modified in manymanners, e.g., increasing the length of two the flanking sequences,replacing the phosphodiester bond with the phosphothioester bond in theoligonucleotide.

The polynucleotide encoding human actin-binding protein 54 can be usedin the diagnosis of human actin-binding protein 54 related diseases. Thepolynucleotide encoding human actin-binding protein 54 can be used todetect whether human actin-binding protein 54 is expressed or not, andwhether the expression of human actin-binding protein 54 is normal orabnormal in the case of diseases. For example, human actin-bindingprotein 54 DNA sequences can be used in the hybridization with biopsysamples to determine the expression of human actin-binding protein 54.The hybridization methods include Southern blotting, Northern blottingand in situ blotting, etc., which are well-known and establishedtechniques. The corresponding kits are commercially available. A part ofor all of the polynucleotides of the invention can be used as probe andfixed on a microarray or DNA chip for analysis of differentialexpression of genes in tissues and for the diagnosis of genes. The humanactin-binding protein 54 specific primers can be used in RNA-polymerasechain reaction and in vitro amplification to detect transcripts of humanactin-binding protein 54.

Further, detection of mutations in human actin-binding protein 54 geneis useful for the diagnosis of human actin-binding protein 54-relateddiseases. Mutations of human actin-binding protein 54 include sitemutation, translocation, deletion, rearrangement and any other mutationscompared with the wild-type human actin-binding protein 54 DNA sequence.The conventional methods, such as Southern blotting, DNA sequencing, PCRand in situ blotting, can be used to detect a mutation. Moreover,mutations sometimes affects the expression of protein. Therefore,Northern blotting and Western blotting can be used to indirectlydetermine whether the gene is mutated or not.

Sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.There is a current need for identifying particular sites of gene on thechromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphism) are presently available for markingchromosomal location. The mapping of DNA to chromosomes according to thepresent invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-35 bp) from the cDNA. These primers are then used for PCRscreening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the oligonucleotideprimers of the invention, sublocalization can be achieved with panels offragments from specific chromosomes or pools of large genomic clones inan analogous manner. Other mapping strategies that can similarly be usedto map to its chromosome include in situ hybridization, prescreeningwith labeled flow-sorted chromosomes and preselection by hybridizationto construct chromosome specific-cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis.

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be the causeof the disease. Comparison of affected and unaffected individualsgenerally involves first looking for structural alterations in thechromosomes, such as deletions or translocations, that are visible fromchromosome level, or detectable using PCR based on that DNA sequence.With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50 to 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

According to the invention, the polypeptides, polynucleotides and itsmimetics, agonists, antagonists and inhibitors may be employed incombination with a suitable pharmaceutical carrier. Such a carrierincludes but is not limited to water, glucose, ethanol, salt, buffer,glycerol, and combinations thereof. Such compositions comprise a safeand effective amount of the polypeptide or antagonist, as well as apharmaceutically acceptable carrier or excipient with no influence onthe effect of the drug. These compositions can be used as drugs indisease treatment.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. With such container(s)there may be a notice from a governmental agency, that regulates themanufacture, use or sale of pharmaceuticals or biological products, thenotice reflects government's approval for the manufacture, use or salefor human administration. In addition, the polypeptides of the inventionmay be employed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner, such as through topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes, humanactin-binding protein 54 is administered in an amount, which iseffective for treating and/or prophylaxis of the specific indication.The amount of human actin-binding protein 54 administrated on patientwill depend upon various factors, such as delivery methods, the subjecthealth, the judgment of the skilled clinician.

1. An isolated polypeptide having an actin-binding protein 54 activityand comprising an amino acid sequence that is at least 95% identical toSEQ ID NO:
 2. 2. The polypeptide of claim 1 wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:
 2. 3. An isolatedpolynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide that has an actin-binding protein54 activity and is at least 95% identical to SEQ ID NO: 2; or (b) apolynucleotide complementary to polynucleotide (a).
 4. A polynucleotideof claim 3 wherein the polynucleotide encodes an amino acid sequence ofSEQ ID NO:2.
 5. A polynucleotide of claim 3 wherein the sequence of saidpolynucleotide comprises position 439-1533 of SEQ ID NO:1.
 6. Apolynucleotide of claim 5 wherein the sequence of said polynucleotidecomprises position 1-1704 of SEQ ID NO:1.
 7. A recombinant vectorcomprising a polynucleotide of claim 3, and a suitable regulatoryelement.
 8. A genetically engineered host cell comprising apolynucleotide of claim
 3. 9. A method for producing a polypeptidehaving an activity of an actin-binding protein 54 which comprises thesteps of: (a) culturing an engineered host cell of claim 8 underconditions suitable for the expression of an actin-binding protein 54;and (b) isolating a polypeptide having the activity of an actin-bindingprotein 54 from the culture.
 10. A composition comprising a polypeptideaccording to claim 1, and a pharmaceutically acceptable carrier.
 11. Acomposition comprising a polynucleotide according to claim 3, and apharmaceutically acceptable carrier.